At the onset of transcription, RNA Polymerase (RNAP) binds to a promoter and causes local strand separation around the start site so that RNA synthesis can begin. The formation of this "open" promoter complex by E. coli RNAP holoenzyme does not require any other protein factor or energy through nucleotide hydrolysis. At present, little is known about the mechanism by which RNAP is able to disrupt base pairing under conditions that greatly favor the duplex structure. The main goal of this project is to understand the mechanics of strand separation, its relationship to the promoter architecture and the function of the initiator protein sigma in this process. Based on a detailed mechanism proposed previously, Dr. deHaseth wishes to determine (a) where the strand separation process is nucleated, (b) whether there are distinct intermediates in the pathway or whether the strand separation is an all-or-none process, and (c) how the duplex is primed for strand separation. Two methods are proposed to capture intermediates in the strand separation process. First, in vitro reactions will be carried out at lower temperatures, a condition previously known to impede strand separation. Second, mutant forms of RNAP holoenzyme will be utilized which bear substitutions of specific amino acids in a region of the sigma factor that has been implicated in strand separation. To pinpoint the sites of nucleation, the kinetics of open complex formation will be measured with promoters that have been engineered to contain bubbles at different positions in and around the region of strand opening. Presumably, a pre-existing helix defect at the site of nucleation would enhance the rate of strand separation. Productive binding of RNAP holoenzyme to the -35 and -10 hexamers is thought to result in the rotation and distortion of the DNA helix. To probe if this plays a role in the nucleation of open complex formation, Dr. deHaseth will examine novel promoters that contain an extended -10 element but not an adequate -35 hexamer sequence. Regions of distortion in these promoters revealed by chemical footprinting will be compared to those at the canonical promoters to determine if anchoring at upstream or downstream sites play a role in open complex formation. An effort will be made to develop a spectrophotometric assay for strand separation by incorporating in this region a fluorescent base, 2 aminopurine. If successful, it will provide a convenient and sensitive method for the kinetic analysis and the characterization of reaction intermediates. Toward a further understanding of promoter structure-function, another goal of the project is to investigate the basis of interference between polymerases bound at divergent promoters. Such a situation is found in many lambdoid phages, where divergent promoters, namely pR and pRM, that dictate the lytic and lysogenic pathway of phage development, are in close proximity to each other and are subject to interference. Dr. deHaseth wishes to precisely delineate the range of inter-promoter distances that cause interference and preclude interference, by deletions and insertions. He will also determine the domains of RNAP that are involved in sensing the presence of an adjacent polymerase, by examining interference with mutant forms of RNA polymerase which bear substitutions and deletions in their alpha and sigma subunits.